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EC number: 264-267-7 | CAS number: 63484-12-8
- Life Cycle description
- Uses advised against
- Endpoint summary
- Appearance / physical state / colour
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- Boiling point
- Density
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- Endpoint summary
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- Ecotoxicological Summary
- Aquatic toxicity
- Endpoint summary
- Short-term toxicity to fish
- Long-term toxicity to fish
- Short-term toxicity to aquatic invertebrates
- Long-term toxicity to aquatic invertebrates
- Toxicity to aquatic algae and cyanobacteria
- Toxicity to aquatic plants other than algae
- Toxicity to microorganisms
- Endocrine disrupter testing in aquatic vertebrates – in vivo
- Toxicity to other aquatic organisms
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- Toxicological Summary
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Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
In vitro gene mutation study in bacteria: Key study: According to OECD 471 Guideline. GLP study. Test item do not induce any mutagenic change in tested bacteria (Salmonella typhimurium TA98, TA100, TA 1535 and TA 1537) and Escherichia coli WP2) under the test conditions.
Link to relevant study records
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- October 26, 2016 - February 22, 2017
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- bacterial reverse mutation assay
- Specific details on test material used for the study:
- STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature (15-25ºC)
- Stability under test conditions: Stable under normal storage condition.
- Solubility and stability of the test substance in the solvent/vehicle: soluble in DMSO - Target gene:
- Histidine-requiring gene in Salmonella typhimurium, Tryptophan-requiring gene for Escherichia coli.
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Additional strain / cell type characteristics:
- other: ∆ uvr B, rfa mutated
- Species / strain / cell type:
- E. coli WP2 uvr A pKM 101
- Additional strain / cell type characteristics:
- other: ∆ uvr A mutated
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 fraction prepared from Sprague Dawley rat liver homogenate
- Test concentrations with justification for top dose:
- Tested doses of test item diluted in DMSO: 50,150,500,1500,5000 ug/plate.
The preliminary study showed no cytotoxicity of the test item at the highest dose tested (5000 μg/plate); therefore this concentrations range was used for the mutagenicity test. - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: It does not react with test item and is compatible with the survival of the bacteria and S9 activity. - Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO, Acetone , NaCl 0.15M
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 7,12-dimethylbenzanthracene
- 9-aminoacridine
- 2-nitrofluorene
- sodium azide
- other: cis-Platinum (II) Diammine Dichloride and 2-Anthramine (see remarks)
- Details on test system and experimental conditions:
- METHOD OF APPLICATION:
ASSAY 1: Plate incorporation method. A first experiment of genotoxicity was conducted, with and without metabolic activation, on the range of concentrations defined by the preliminary study: 5000µg, 1500µg, 500µg, 150µg, and 50µg dose/plate.
Salmonella Typhimurium strains: for each strain, 0.1 mL of the bacterial suspension containing 1-9 x109 bacteria/mL and 0.1 mL of each dilution of the original solution and 0.5 mL of sterile phosphate buffer are successively added to 2 mL of overlay agar, maintained supercooled at 45° C, containing 10 % (v/v) of a L-Histidine-D-Biotine solution (0.5 mM).
Escherichia coli strain : in a test tube 0.1 mL of the bacterial suspension containing 1-9 x 109 bacteria/mL and 0.1 mL of each dilution of the original solution and 0.5 mL of phosphate buffer are successively added to 2 mL of overlay agar maintained super cooled in 45° C containing 5% (v/v) of nutrient broth n° 2 to which are added 5 μL of a L-Tryptophane solution at 2 mg/mL.
For the assay with metabolic activation 500 μL of S9-mix fraction were quickly added, before pouring the mixture onto the plates.
Afterwards, plates were incubated at 37° C over a 48-72 hour period. The number of revertant colonies per plate was counted.
ASSAY 2: Preincubation method: a second experiment was performed, also with and without metabolic activation, with dose levels defined after analysis of results obtained on the first experiment. This second experiment was performed in order to confirm or for complement results of the first one. The protocol is similar to the Assay 1, except for that the solution of the test item solution with the test strain are preincubated with shaking for 30 min., at 37° C prior to mixing with the overlay agar and pouring onto the minimal agar plate. When metabolic activation is used, 500 μL of S9-mix fraction are added before the pre-incubation.
- Cell density at seeding: 1.9E09 bacteria/mL
DURATION
- Preincubation period: 30min at 37ºC, only for the Test 2.
- Exposure duration: 48-72h
SELECTION AGENT: absence of Histidin and Tryptophan, the lack of amino-acid in the medium. Only the mutants can grow due to their capability to synthesize the essential amino acid.
NUMBER OF REPLICATIONS: 3
DETERMINATION OF CYTOTOXICITY: Cytotoxicity may be detected by a reduction in the number of revertant colonie.s.
A preliminary cytotoxicity test was performed with the strain TA100 to determine any sign of cytotoxicity. The doses used were 50µg, 150µg, 500µg, 1500µg, 2500µg and 5000µg dose/plate.
In a test tube 0.1 mL of the bacterial suspension (1-9 x 103 bacteria/mL) and 0.1 mL of the stock solution and dilutions, are successively added to 2 mL of top agar at 45°C, containing 10 % (v/v) of a solution of L-Histidine-D-Biotine (2.5 mM). After homogenization, the content of the tube is poured onto a Petri plate (90 mm in diameter) containing minimal agar (20 mL). 3 plates per concentration are incubated for 48-72 hours at 37°C, and the colonies counted. Petri plates were observed and any cytotoxicity sign was reported (bottom bacterial layer reduction). A negative control containing the blank alone is run in parallel.In case bacteriostatic activity is detected, the highest concentration to be retained is that exhibiting a bacteriostatic activity of 75 % or less. The precipitate, if present, should not interfere with the scoring.
OTHER EXAMINATIONS:
Test item and the corresponding dilutions are added to 2 mL of top agar maintained at 45°C, and poured after homogenization on the bottom agar (20 ml) onto a Petri plate (90 mm in diameter) (n = 3). Plates are incubated for 48 - 72 hours at 37°C and then examined. There should be no bacterial growth on any plate. S9-mix sterility is checked using the same protocol. - Rationale for test conditions:
- Test conditions according to the OECD nº471. Dose selection for main assays were perfromed based on results of the preliminary cytotoxicity test.
- Evaluation criteria:
- The result of the test is considered as negative if the revertant number is below three fold the number of spontaneous reversions, for TA 1535 and TA 1537 strains, and below two fold the number of spontaneous reversions for TA 98, TA 100 and Escherichia coli WP2(uvrA-) (pKM 101) strains without and with metabolic activation.
The result of the test is considered positive if a dependent relationship concentration is obtained in one, or several of the 5 strains, without and/or with metabolic activation, a mutagenic effect being taken into account for a given dilution of test item if the number of revertant colonies is at least two fold that of spontaneous revertant colonies for TA 98, TA 100 and Escherichia coli WP2(uvrA-) (pKM 101), and three fold for TA 1535 and TA 1537.
All results must be confirmed in an independent experiment. - Key result
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- E. coli WP2 uvr A pKM 101
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: no precipitation of the test item was observed.
RANGE-FINDING/SCREENING STUDIES:
Preliminary study was carried out with S. thyphymurium TA 100 with doses up to 5000µg/plate. Neither original solution nor dilutions have bacteriostatic effect. For this reason, on the Assay 1, the following doses were used: 5 000, 1 500, 500, 150 and 50 μg/plate.
HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive control data
Without metabolic activation:
TA 1535 N=912,Mean=668.2±202.4 (min/max=45.0/1481.0),
TA 1537 N=912,Mean=787,5±385.0 (min/max=219.0/1967.0)
TA 98 N=912,Mean=532.2±214.4 (min/max=187.0/1667.0)
TA 100 N=912,Mean=862.4±270.3 (min/max=381.0-1690.0)
Escherichia coli WP2(pKM101) (uvrA-) N=675, Mean=531.1±160.5 (min/max=280.0/1089.0)
With metabolic activation - without pre-incubation
TA 1535 N=492,Mean=95.2±46.9 (min/max=26.0/269.0)
TA 1537 N=492,Mean=53.6±21.7 (min/max=24.0/170.0)
TA 98 N=492,Mean=566.3±195.1 (min/max=219.0/1280.0)
TA 100 N=489,Mean=898.1±374.0 (min/max=361.0/2163.0)
Escherichia coli WP2(pKM101) (uvrA-) N=351,Mean=739.1±234.7 (min/max=384.0/1680.0)
With metabolic activation - with pre-incubation
TA 1535 N=489,Mean=69.9±29.2 (min/max=25.0/185.0),
TA 1537 N=489,Mean=69.9±29.2 (min/max=21.0/182.0),
TA 98 N=489,Mean=479.4±183.9(min/max=174.0/1370.0),
TA 100 N=492,Mean=708.0±295.9(min/max=309.0/1889.0)
Escherichia coli WP2(pKM101) (uvrA-)N=351,Mean=731.7±217.3 (min/max=405.0/1680.0)
- Negative (solvent/vehicle) historical control data:
Without metabolic activation:
TA 1535 N=912,Mean=10.7±3.4 (min/max=4.0/23.0)
TA 1537 N=912,Mean=5.8±2.4 (min/max=1.0/14.0)
TA 98 N=912,Mean=15.7±3.7 (min/max=6.0/29.0)
TA 100 N=912,Mean=65.2±17.1(min/max=41.0/158.0)
Escherichia coli WP2(pKM101) (uvrA-) N=675,Mean=67.8±20.0(min/max=40.0-/65.0)
With metabolic activation - without pre-incubation
TA 1535 N=492,Mean=12.2±4.0 (min/max=3.0/23.0)
TA 1537 N=492,Mean 7.9±3.0, (min/max=1.0/24.0)
TA 98 N=492,Mean=22.9±4.6 (min/max=12.0/35.0)
TA 100 N=489,Mean=108.3±25.1 (min/max=58.0/220.0),
Escherichia coli WP2(pKM101) (uvrA-) N=351,Mean=153.1±28.9 (min/max=80.0/264.0)
With metabolic activation, with pre-incubation
TA 1535 N=489,Mean=12.6±4.1 (min/max=5.0/25.0),
TA 1537 N=489,Mean=8.2±3.0(min/max=2.0/19.0)
TA 98 N=489,Mean=23.0±4.9(min/max=11.0/35.0)
TA 100 N=489,Mean=108.4±27.1(min/max=51.0/217.0)
Escherichia coli WP2(pKM101) (uvrA-) N=351,Mean=158.5±31.4 (min/max=69.0/250.0) - Conclusions:
- Test item do not induce any mutagenic change in tested bacterias (Salmonella typhimurium TA98, TA100, TA 1535 and TA 1537) and Escherichia coli WP2 (uvrA-) (pKM101) with and without metabolic activation up to 5000 μg/plate.
- Executive summary:
The aim of this test was to determine the ability of the test item to induce mutation, it was assessed using the bacterial reverse mutation test (Ames test). The test was performed following the OECD Guideline 471 with GLP on four Salmonella typhimurium strains and on Escherichia coli WP2. The test item dilutions were prepared in DMSO for analysis. Firstly, a preliminary cytotoxicity test was performed on S. typhimurium TA100 strain with the following concentrations: 5 000, 2500, 1 500, 500, 150 and 50μg/plate, for triplicate. Plates were incubated at 37ºC for 48-72h and after this period colonies were counted. As the preliminary study showed no cytotoxicity of the test item, the concentrations 5 000, 1 500, 500, 150 and 50 μg/plate were used for the genotoxicity Test 1 (direct method) and Test 2 (pre-incubation method), and positive and negative/solvent controls were included. The same test conditions as the preliminary test were used, with and without metabolic activation system S9 mix. The revertant analysis showed no cytotoxic effect. There was no evidence of any increase in the number of revertant colonies in the presence of the test item stock solution and dilutions without and with metabolic activation. Based on the result of this study, the test item was not found to be mutagenic under the test conditions.
Reference
Table No.6: Sterility control:
Serie |
Doses |
Colony number/plate |
||
Control nº1 |
|
1 |
2 |
3 |
Solution of test item Lemi code :GFF211116-S2 |
5000ug/plate |
0 |
0 |
0 |
1500ug/plate |
0 |
0 |
0 |
|
500ug/plate |
0 |
0 |
0 |
|
150ug/plate |
0 |
0 |
0 |
|
50ug/plate |
0 |
0 |
0 |
|
S9-mix |
500ul/plate |
0 |
0 |
0 |
Control nº2 |
|
1 |
2 |
3 |
Solution of test item Lemi code :GFF281116-S2 |
5000ug/plate |
0 |
0 |
0 |
1500ug/plate |
0 |
0 |
0 |
|
500ug/plate |
0 |
0 |
0 |
|
150ug/plate |
0 |
0 |
0 |
|
50ug/plate |
0 |
0 |
0 |
|
S9-mix |
500ul/plate |
0 |
0 |
0 |
Table No.7: Bacteriostatic activity control (nº1 and nº2)
|
|
0 Negative control |
DMSO |
50ug |
150ug |
500ug |
1500ug |
2500ug |
5000ug |
Solution of test item Lemi code :GFF211116-S2 |
N1 |
249 |
236 |
249 |
255 |
264 |
249 |
238 |
261 |
N2 |
265 |
248 |
258 |
265 |
258 |
243 |
251 |
246 |
|
N3 |
259 |
249 |
245 |
257 |
246 |
249 |
263 |
240 |
|
N |
258+-8 |
244+-7 |
251+-7 |
259+-5 |
256+-9 |
247+-3 |
251+-13 |
249+-11 |
|
% |
- |
95 |
97 |
101 |
99 |
96 |
97 |
97 |
|
|
|
0 Negative control |
DMSO |
50ug |
150ug |
500ug |
1500ug |
5000ug |
|
Solution of test item Lemi code :GFF281116-S2 |
N1 |
231 |
265 |
237 |
276 |
223 |
231 |
223 |
|
N2 |
281 |
285 |
228 |
260 |
242 |
264 |
261 |
|
|
N3 |
272 |
225 |
252 |
249 |
261 |
275 |
267 |
|
|
N |
261+-27 |
258+-31 |
239+-12 |
262+-14 |
242+-19 |
257+-23 |
250+-24 |
|
|
% |
- |
99 |
91 |
100 |
93 |
98 |
96 |
|
N1:number of colonies in plate 1
N2:number of colonies in plate 2
N3:number of colonies in plate 3
N:mean per plate
%: Percent of survival compared to negative control
Table No.8: TA 1535 Assay nº1- without metabolic activation (-S9 -mix)
Serie |
Dose/plate |
Nº1 |
Nº2 |
N3 |
mean |
Standart deviation |
R |
Negative control |
100uL |
14 |
14 |
14 |
14.00 |
0.00 |
- |
Positive control solvent |
5uL |
15 |
7 |
15 |
12.33 |
4.62 |
- |
Positive control Sodium azide |
5ug In 5uL |
740 |
561 |
696 |
665.67 |
93.28 |
53.97 |
Vehicle |
50uL |
17 |
7 |
12 |
12.00 |
5.00 |
- |
Solution of test item Lemi code :GFF211116-S2 |
5000ug/plate |
10 |
4 |
12 |
8.67 |
4.16 |
0.72 |
1500ug/plate |
11 |
10 |
13 |
11.33 |
1.53 |
0.94 |
|
500ug/plate |
8 |
18 |
9 |
11.67 |
5.51 |
0.97 |
|
150ug/plate |
8 |
11 |
10 |
9.67 |
1.53 |
0.81 |
|
50ug/plate |
7 |
15 |
13 |
11.67 |
4.16 |
0.97 |
Table No.9: TA 1535 Assay nº1- with metabolic activation (10% S9 -mix) - without preincubation
Serie |
Dose/plate |
Nº1 |
Nº2 |
Nº3 |
mean |
Standart deviation |
R |
Negative control |
100uL |
21 |
5 |
18 |
14.67 |
8.50 |
- |
Positive control solvent |
20uL |
9 |
11 |
9 |
9.67 |
1.15 |
- |
Positive control 2-Anthramide |
2ug In 20uL |
152 |
161 |
128 |
147.00 |
17.06 |
15.21 |
Vehicle |
50uL |
18 |
9 |
15 |
14.00 |
4.58 |
- |
Solution of test item Lemi code :GFF211116-S2 |
5000ug/plate |
9 |
12 |
8 |
9.67 |
2.08 |
0.69 |
1500ug/plate |
14 |
12 |
13 |
13.00 |
1.00 |
0.93 |
|
500ug/plate |
14 |
15 |
13 |
14.00 |
1.00 |
1.00 |
|
150ug/plate |
7 |
15 |
8 |
10.00 |
4.36 |
0.71 |
|
50ug/plate |
14 |
9 |
12 |
11.67 |
2.52 |
0.83 |
Table No.10: TA 1535Assay nº2- without metabolic activation (-S9 -mix)
Serie |
Dose/plate |
Nº1 |
Nº2 |
Nº3 |
mean |
Standart deviation |
R |
Negative control |
100uL |
10 |
13 |
15 |
12.67 |
2.52 |
- |
Positive control solvent |
5uL |
8 |
9 |
7 |
8.00 |
1.00 |
- |
Positive control Sodium Azide |
5ug In 5uL |
743 |
759 |
772 |
758 |
14.53 |
94.75 |
Vehicle |
50uL |
16 |
12 |
11 |
13.00 |
2.65 |
- |
Solution of test item Lemi code :GFF281116-S2 |
5000ug/plate |
11 |
8 |
13 |
10.67 |
2.52 |
0.82 |
1500ug/plate |
12 |
14 |
7 |
11.00 |
3.61 |
0.85 |
|
500ug/plate |
11 |
16 |
14 |
13.67 |
2.52 |
1.05 |
|
150ug/plate |
6 |
11 |
14 |
10.33 |
4.04 |
0.79 |
|
50ug/plate |
15 |
8 |
18 |
13.67 |
5.13 |
1.05 |
Table No.11: TA 1535 Assay nº2- with metabolic activation (10% S9 -mix) - with preincubation
Serie |
Dose/plate |
Nº1 |
Nº2 |
Nº3 |
mean |
Standart deviation |
R |
Negative control |
100uL |
12 |
12 |
17 |
13.67 |
2.89 |
- |
Positive control solvent |
10uL |
10 |
11 |
9 |
10.00 |
1.00 |
- |
Positive control 2-Anthramide |
1ug In 10uL |
52 |
65 |
45 |
54.00 |
10.15 |
5.40 |
Vehicle |
50uL |
13 |
12 |
16 |
13.67 |
2.08 |
- |
Solution of test item Lemi code :GFF281116-S2 |
5000ug/plate |
12 |
10 |
10 |
10.67 |
1.15 |
0.78 |
1500ug/plate |
8 |
16 |
8 |
10.67 |
4.62 |
0.78 |
|
500ug/plate |
16 |
10 |
13 |
13.00 |
3.00 |
0.95 |
|
150ug/plate |
17 |
15 |
8 |
13.33 |
4.73 |
0.98 |
|
50ug/plate |
15 |
12 |
13 |
13.33 |
1.53 |
0.98 |
Table No.12 TA1537 Assay nº1 -without metabolic activation (-S9 -mix)
Serie |
Dose/plate |
Nº1 |
Nº2 |
Nº3 |
mean |
Standart deviation |
R |
Negative control |
100uL |
2 |
8 |
6 |
5.33 |
3.06 |
- |
Positive control solvent |
20uL |
3 |
5 |
3 |
3.67 |
1.15 |
- |
Positive control 9-Aminoacridine |
50ug In 20uL |
1563 |
1673 |
1645 |
1627.00 |
57.17 |
443.73 |
Vehicle |
50uL |
6 |
6 |
5 |
5.33 |
0.58 |
- |
Solution of test item Lemi code :GFF281116-S2 |
5000ug/plate |
5 |
5 |
9 |
6.67 |
2.08 |
1.25 |
1500ug/plate |
8 |
12 |
4 |
8.00 |
4.00 |
1.50 |
|
500ug/plate |
6 |
9 |
5 |
6.67 |
2.08 |
1.25 |
|
150ug/plate |
5 |
5 |
3 |
4.33 |
1.15 |
0.81 |
|
50ug/plate |
3 |
7 |
5 |
5.00 |
2.00 |
0.94 |
Table No.13 TA1537 Assay nº1 -with metabolic activation (10% -S9 -mix) - without preincubation
Serie |
Dose/plate |
Nº1 |
Nº2 |
Nº3 |
mean |
Standart deviation |
R |
Negative control |
100uL |
16 |
12 |
19 |
15.67 |
3.51 |
- |
Positive control solvent |
20uL |
12 |
11 |
12 |
11.67 |
0.58 |
- |
Positive control 2-Anthramine |
2ug In 20uL |
43 |
72 |
63 |
59.33 |
14.84 |
5.09 |
Vehicle |
50uL |
11 |
10 |
13 |
11.33 |
1.53 |
- |
Solution of test item Lemi code :GFF281116-S2 |
5000ug/plate |
8 |
10 |
5 |
7.67 |
2.52 |
0.68 |
1500ug/plate |
14 |
10 |
9 |
11.00 |
2.65 |
0.97 |
|
500ug/plate |
7 |
8 |
14 |
9.67 |
3.79 |
0.85 |
|
150ug/plate |
6 |
14 |
7 |
9.00 |
4.36 |
0.79 |
|
50ug/plate |
4 |
13 |
14 |
10.33 |
5.51 |
0.91 |
Table No.14 TA1537 Assay nº2 -without metabolic activation (-S9 -mix)
Serie |
Dose/plate |
Nº1 |
Nº2 |
Nº3 |
mean |
Standart deviation |
R |
Negative control |
100uL |
4 |
2 |
5 |
3.67 |
1.53 |
- |
Positive control solvent |
20uL |
3 |
7 |
11 |
7.00 |
4.00 |
- |
Positive control 9-Aminoacridine |
50ug In 20uL |
1581 |
727 |
1591 |
1299.67 |
495.97 |
185.67 |
Vehicle |
50uL |
11 |
11 |
13 |
11.67 |
1.15 |
- |
Solution of test item Lemi code :GFF281116-S2 |
5000ug/plate |
6 |
9 |
4 |
6.33 |
2.52 |
0.54 |
1500ug/plate |
5 |
5 |
6 |
5.33 |
0.58 |
0.46 |
|
500ug/plate |
11 |
8 |
3 |
7.33 |
4.04 |
0.63 |
|
150ug/plate |
6 |
6 |
6 |
6.00 |
0.00 |
0.51 |
|
50ug/plate |
10 |
4 |
8 |
7.33 |
3.06 |
0.63 |
Table No.15 TA1537 Assay nº2 -with metabolic activation (10%-S9 -mix) - with preincubation
Serie |
Dose/plate |
Nº1 |
Nº2 |
Nº3 |
mean |
Standart deviation |
R |
Negative control |
100uL |
10 |
14 |
11 |
11.67 |
2.08 |
- |
Positive control solvent |
10uL |
8 |
9 |
10 |
9.00 |
1.00 |
- |
Positive control 2-Anthramine |
1ug In 10uL |
27 |
31 |
40 |
32.67 |
6.66 |
3.63 |
Vehicle |
50uL |
8 |
12 |
16 |
12.00 |
4.00 |
- |
Solution of test item Lemi code :GFF281116-S2 |
5000ug/plate |
5 |
9 |
13 |
9.00 |
4.00 |
0.75 |
1500ug/plate |
13 |
6 |
19 |
12.67 |
6.51 |
1.06 |
|
500ug/plate |
10 |
13 |
10 |
11.00 |
1.73 |
0.92 |
|
150ug/plate |
11 |
10 |
10 |
10.33 |
0.58 |
0.86 |
|
50ug/plate |
12 |
9 |
7 |
9.33 |
2.52 |
0.78 |
Table No.16 TA98 Assay nº1- without metabolic activation (-S9 -mix)
Serie |
Dose/plate |
Nº1 |
Nº2 |
Nº3 |
mean |
Standart deviation |
R |
Negative control |
100uL |
18 |
11 |
18 |
15.67 |
4.04 |
- |
Positive control solvent |
20uL |
18 |
10 |
17 |
15.00 |
4.36 |
- |
Positive control 2-Nitrofluorene |
2ug In 20uL |
286 |
268 |
298 |
284.00 |
15.10 |
18.93 |
Vehicle |
50uL |
12 |
15 |
16 |
14.33 |
2.08 |
- |
Solution of test item Lemi code :GFF211116-S2 |
5000ug/plate |
15 |
11 |
16 |
14.00 |
2.65 |
0.98 |
1500ug/plate |
12 |
11 |
15 |
12.67 |
2.08 |
0.88 |
|
500ug/plate |
11 |
14 |
22 |
15.67 |
5.69 |
1.09 |
|
150ug/plate |
15 |
17 |
11 |
14.33 |
3.06 |
1.00 |
|
50ug/plate |
16 |
24 |
19 |
19.67 |
4.04 |
1.37 |
Table No.17 TA98 Assay nº1- with metabolic activation (10% -S9 -mix)- without preincubation
Serie |
Dose/plate |
Nº1 |
Nº2 |
Nº3 |
mean |
Standart deviation |
R |
Negative control |
100uL |
18 |
21 |
25 |
21.33 |
3.51 |
- |
Positive control solvent |
20uL |
23 |
16 |
19 |
19.33 |
3.51 |
- |
Positive control 2-Anthramine |
2ug In 20uL |
442 |
429 |
293 |
388.00 |
82.53 |
20.07 |
Vehicle |
50uL |
19 |
18 |
12 |
16.33 |
3.79 |
- |
Solution of test item Lemi code :GFF211116-S2 |
5000ug/plate |
16 |
15 |
22 |
17.67 |
3.79 |
1.08 |
1500ug/plate |
31 |
19 |
19 |
23.00 |
6.93 |
1.41 |
|
500ug/plate |
21 |
24 |
17 |
20.67 |
3.51 |
1.27 |
|
150ug/plate |
12 |
12 |
23 |
15.67 |
6.35 |
0.96 |
|
50ug/plate |
23 |
22 |
24 |
23.00 |
1.00 |
1.41 |
Table No.18 TA98 Assay nº2- without metabolic activation (-S9 -mix)
Serie |
Dose/plate |
Nº1 |
Nº2 |
Nº3 |
mean |
Standart deviation |
R |
Negative control |
100uL |
15 |
11 |
18 |
14.67 |
3.51 |
- |
Positive control solvent |
20uL |
12 |
18 |
17 |
15.67 |
3.21 |
- |
Positive control 2-Nitrofluorene |
2ug In 20uL |
302 |
198 |
195 |
231.67 |
60.93 |
14.79 |
Vehicle |
50uL |
22 |
18 |
19 |
19.67 |
2.08 |
- |
Solution of test item Lemi code :GFF281116-S2 |
5000ug/plate |
9 |
14 |
11 |
11.33 |
2.52 |
0.58 |
1500ug/plate |
15 |
15 |
15 |
15.00 |
0.00 |
0.76 |
|
500ug/plate |
11 |
17 |
15 |
14.33 |
3.06 |
0.73 |
|
150ug/plate |
14 |
25 |
17 |
18.67 |
5.69 |
0.95 |
|
50ug/plate |
16 |
16 |
21 |
17.67 |
2.89 |
0.90 |
Table No.19 TA98 Assay nº2- with metabolic activation (10%-S9 -mix) - with preincubation
Serie |
Dose/plate |
Nº1 |
Nº2 |
Nº3 |
mean |
Standart deviation |
R |
Negative control |
100uL |
28 |
30 |
20 |
26.00 |
5.29 |
- |
Positive control solvent |
10uL |
17 |
11 |
13 |
17.00 |
8.73 |
- |
Positive control 2-Anthramine |
1ug In 10uL |
178 |
199 |
203 |
193.33 |
13.43 |
11.37 |
Vehicle |
50uL |
27 |
22 |
21 |
23.33 |
3.21 |
- |
Solution of test item Lemi code :GFF281116-S2 |
5000ug/plate |
29 |
28 |
22 |
26.33 |
3.79 |
1.13 |
1500ug/plate |
18 |
28 |
20 |
22.00 |
5.29 |
0.94 |
|
500ug/plate |
22 |
20 |
23 |
21.67 |
1.53 |
0.93 |
|
150ug/plate |
27 |
21 |
30 |
26.00 |
4.58 |
1.11 |
|
50ug/plate |
23 |
32 |
29 |
28.00 |
4.58 |
1.20 |
Table No.20 TA 100 Assay nº1- without metabolic activation (-S9 -mix)
Serie |
Dose/plate |
Nº1 |
Nº2 |
Nº3 |
mean |
Standart deviation |
R |
Negative control |
100uL |
49 |
41 |
64 |
51.33 |
11.68 |
- |
Positive control solvent |
20uL |
50 |
60 |
60 |
56.67 |
5.77 |
- |
Positive control Sodium Azide |
20ug In 20uL |
1646 |
1510 |
1589 |
1581.67 |
68.30 |
27.91 |
Vehicle |
50uL |
72 |
76 |
74 |
74.00 |
2.00 |
- |
Solution of test item Lemi code :GFF211116-S2 |
5000ug/plate |
81 |
50 |
46 |
59.00 |
19.16 |
0.80 |
1500ug/plate |
63 |
81 |
65 |
69.67 |
9.87 |
0.94 |
|
500ug/plate |
52 |
71 |
54 |
59.00 |
10.44 |
0.80 |
|
150ug/plate |
66 |
68 |
73 |
69.00 |
3.61 |
0.93 |
|
50ug/plate |
75 |
73 |
70 |
72.67 |
2.52 |
0.98 |
Table No.21 TA 100 Assay nº1- with metabolic activation (10% -S9 -mix) without preincubation
Serie |
Dose/plate |
Nº1 |
Nº2 |
Nº3 |
mean |
Standart deviation |
R |
Negative control |
100uL |
72 |
91 |
69 |
76.67 |
12.42 |
- |
Positive control solvent |
20uL |
72 |
77 |
76 |
75.00 |
2.65 |
- |
Positive control 2-Anthramine |
2ug In 20uL |
746 |
563 |
555 |
621.33 |
108.04 |
8.28 |
Vehicle |
50uL |
72 |
73 |
74 |
73.00 |
1.00 |
- |
Solution of test item Lemi code :GFF211116-S2 |
5000ug/plate |
91 |
82 |
72 |
81.67 |
9.50 |
1.12 |
1500ug/plate |
71 |
72 |
71 |
71.33 |
0.58 |
0.98 |
|
500ug/plate |
78 |
84 |
72 |
78.00 |
6.00 |
1.07 |
|
150ug/plate |
101 |
82 |
98 |
93.67 |
10.21 |
1.28 |
|
50ug/plate |
79 |
77 |
75 |
77.00 |
2.00 |
1.05 |
Table No.22 TA 100 Assay nº2- without metabolic activation (-S9 -mix)
Serie |
Dose/plate |
Nº1 |
Nº2 |
Nº3 |
mean |
Standart deviation |
R |
Negative control |
100uL |
59 |
76 |
112 |
82.33 |
27.06 |
- |
Positive control solvent |
20uL |
59 |
71 |
60 |
63.33 |
6.66 |
- |
Positive control Sodium Azide |
20ug In 20uL |
1622 |
1274 |
1561 |
1485.67 |
185.83 |
23.46 |
Vehicle |
50uL |
73 |
66 |
71 |
70.00 |
3.61 |
- |
Solution of test itemLemi code :GFF281116-S2 |
5000ug/plate |
73 |
62 |
64 |
66.33 |
5.86 |
0.95 |
1500ug/plate |
57 |
57 |
68 |
60.67 |
6.35 |
0.87 |
|
500ug/plate |
78 |
71 |
62 |
70.33 |
8.02 |
1.00 |
|
150ug/plate |
72 |
61 |
77 |
70.00 |
8.19 |
1.00 |
|
50ug/plate |
61 |
68 |
68 |
65.67 |
4.04 |
0.94 |
Table No.23 TA 100 Assay nº2- without metabolic activation (10% -S9 -mix) with preincubation
Serie |
Dose/plate |
Nº1 |
Nº2 |
Nº3 |
mean |
Standart deviation |
R |
Negative control |
100uL |
84 |
79 |
62 |
75.00 |
11.53 |
- |
Positive control solvent |
10uL |
79 |
86 |
70 |
78.33 |
8.02 |
- |
Positive control 2-Anthramine |
1ug In 10uL |
544 |
426 |
561 |
510.33 |
73.53 |
6.51 |
Vehicle |
50uL |
54 |
68 |
59 |
60.33 |
7.09 |
- |
Solution of test item Lemi code :GFF281116-S2 |
5000ug/plate |
65 |
85 |
72 |
74.00 |
10.15 |
1.23 |
1500ug/plate |
80 |
84 |
65 |
76.33 |
10.02 |
1.27 |
|
500ug/plate |
79 |
107 |
97 |
94.33 |
14.19 |
1.56 |
|
150ug/plate |
93 |
108 |
90 |
97.00 |
9.64 |
1.61 |
|
50ug/plate |
87 |
75 |
92 |
84.67 |
8.74 |
1.40 |
Table No.24 E.coli Assay nº1- without metabolic activation (-S9 -mix)
Serie |
Dose/plate |
Nº1 |
Nº2 |
Nº3 |
mean |
Standart deviation |
R |
Negative control |
100uL |
47 |
478 |
59 |
51.33 |
6.66 |
- |
Positive control solvent |
10uL |
48 |
61 |
49 |
52.67 |
7.23 |
- |
Positive control Cis-Platinum (II) |
1ug In 10uL |
280 |
311 |
297 |
296.00 |
15.52 |
5.62 |
Vehicle |
50uL |
129 |
164 |
138 |
143.67 |
18.18 |
- |
Solution of test item Lemi code :GFF211116-S2 |
5000ug/plate |
102 |
24 |
43 |
56.33 |
40.67 |
0.39 |
1500ug/plate |
78 |
78 |
102 |
86.00 |
13.86 |
0.6 |
|
500ug/plate |
77 |
84 |
69 |
76.67 |
7.51 |
0.53 |
|
150ug/plate |
71 |
68 |
74 |
71.00 |
3.00 |
0.49 |
|
50ug/plate |
73 |
124 |
91 |
96.00 |
25.87 |
0.67 |
Table No.25 E.coli Assay nº1- with metabolic activation (10% -S9 -mix) without preincubation
Serie |
Dose/plate |
Nº1 |
Nº2 |
Nº3 |
mean |
Standart deviation |
R |
Negative control |
100uL |
206 |
196 |
216 |
206.00 |
10.00 |
- |
Positive control solvent |
5uL |
217 |
179 |
184 |
193.33 |
20.65 |
- |
Positive control DimethylbenzathracEne |
5ug In 5uL |
428 |
435 |
415 |
426.00 |
10.15 |
2.20 |
Vehicle |
50uL |
215 |
208 |
210 |
211.00 |
3.61 |
- |
Solution of test item Lemi code :GFF211116-S2 |
5000ug/plate |
59 |
131 |
154 |
114.67 |
49.56 |
0.54 |
1500ug/plate |
162 |
149 |
191 |
167.33 |
21.50 |
0.79 |
|
500ug/plate |
154 |
145 |
176 |
158.33 |
15.95 |
0.75 |
|
150ug/plate |
170 |
187 |
183 |
180.00 |
8.89 |
0.85 |
|
50ug/plate |
173 |
184 |
191 |
182.67 |
9.07 |
0.87 |
Table No.26 E.coli Assay nº2- without metabolic activation (-S9 -mix)
Serie |
Dose/plate |
Nº1 |
Nº2 |
Nº3 |
mean |
Standart deviation |
R |
Negative control |
100uL |
91 |
108 |
107 |
102.00 |
9.54 |
- |
Positive control solvent |
10uL |
126 |
133 |
119 |
126.00 |
7.00 |
- |
Positive control Cis-Platinum (II) |
1ug In 10uL |
348 |
327 |
301 |
325.33 |
23.54 |
2.58 |
Vehicle |
50uL |
118 |
133 |
134 |
128.33 |
8.96 |
- |
Solution of test item Lemi code :GFF281116-S2 |
5000ug/plate |
50 |
24 |
79 |
51.00 |
27.51 |
0.40 |
1500ug/plate |
82 |
78 |
53 |
71.00 |
15.72 |
0.55 |
|
500ug/plate |
114 |
97 |
100 |
103.67 |
9.07 |
0.81 |
|
150ug/plate |
132 |
123 |
126 |
127.00 |
4.58 |
0.99 |
|
50ug/plate |
114 |
138 |
115 |
122.33 |
13.58 |
0.95 |
Table No.27 E.coli Assay nº2- with metabolic activation (10% -S9 -mix) with preincubation
Serie |
Dose/plate |
Nº1 |
Nº2 |
Nº3 |
mean |
Standart deviation |
R |
Negative control |
100uL |
161 |
155 |
187 |
167.67 |
17.01 |
- |
Positive control solvent |
5uL |
186 |
179 |
199 |
188.00 |
10.15 |
- |
Positive control DimethylbenzathracEne |
2.5ug In 5uL |
512 |
537 |
428 |
492.33 |
57.10 |
2.62 |
Vehicle |
50uL |
169 |
197 |
188 |
184.67 |
14.29 |
- |
Solution of test item Lemi code :GFF281116-S2 |
5000ug/plate |
124 |
184 |
82 |
130.00 |
51.26 |
0.70 |
1500ug/plate |
136 |
125 |
117 |
126.00 |
9.54 |
0.68 |
|
500ug/plate |
197 |
163 |
186 |
182.00 |
17.35 |
0.99 |
|
150ug/plate |
191 |
184 |
209 |
194.67 |
12.90 |
1.05 |
|
50ug/plate |
184 |
159 |
170 |
171.00 |
12.53 |
0.93 |
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Endpoint conclusion
- Endpoint conclusion:
- no study available
Additional information
Key Study - In vitro gene mutation study in bacteria: According to OECD 471 Guideline. GLP study. The test was performed on four Salmonella typhimurium strains and on Escherichia coli WP2. After a preliminary test which showed no cytotoxicity of the test item, a direct method test and a pre-incubation method test were performed with the doses of 5 000, 1 500, 500, 150 and 50 μg/plate of test item. There was no evidence of any increase in the number of revertant colonies in the presence of the test item stock solution and dilutions without and with metabolic activation. Based on the result of this study, the test item was not found to be mutagenic under the test conditions.
Justification for classification or non-classification
Based on the available information, the test item is not classified as mutagen according to CLP Regulation (EC) no.1272/2008.
Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.
Reproduction or further distribution of this information may be subject to copyright protection. Use of the information without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner.